Methods of Determination of Pathogenic Microbes. 113 



This method is therefore useful for determining the 

 presence of microbes in any tissue. In its execution 

 an equal number of sections are treiited bj both 

 methods. 



1. Method by Kilhne's blue. The blue of Kilhne is 

 obtained by adding, drop by drop, to a one per cent 

 solution of carbonate of ammonia, a saturated aque- 

 ous solution of methylene blue until the mixture ac- 

 quires a deep blue color. The sections, after dehy- 

 dration in alcohol, remain ten minutes in this blue 

 liquid, are then rapidly decolorized (two or three sec- 

 onds) in a 1 to 1000 aqueous solution of hydrochloric 

 acid, then passed into distilled water to remove all 

 trace of the acid. They are afterward mounted with- 

 out passing through alcohol, which would remove 

 too much of the stain. For this end, after removal 

 from the water, they are spread out on a slide and 

 there allowed to dry, or better, the desiccation is has- 

 tened either by means of a current of air or very 

 slight heating. They are then cleared in xylol and 

 mounted in balsam. 



2. Method by crystal violet. — Kilhne's violet is obtained 

 in the same way as the blue solution by adding sev- 

 eral drops of a saturated aqueous solution of crystal 

 violet to a one per cent aqueous solution of carbonate 

 of ammonia until a deep violet color is produced. 

 Sections, after dehydration in alcohol, are left in this 

 solution ten minutes (two hours for the bacillus tuber- 

 culosis), washed in distilled water, and immersed for 

 two to three minutes in Gram's iodine solution ; from 

 this they are transferred into a saturated alcoholic 

 solution of fluorescine until decoloration is nearly 

 complete. The remaining coloring matter is extracted 



